1. Field of the Invention
This invention relates to a three-dimensional confocal microscope system and particularly to improvements made to correct brightness of a confocal image in the direction of depth of a sample and carry out three-dimensional observation of the sample with accurate brightness.
2. Description of the Related Art
With a confocal microscope, it is possible to acquire sliced images of a sample without thinly segmenting the sample and to construct a precise three-dimensional image of the sample from the sliced images. Therefore, the confocal microscope is used for observation of physiological reactions and morphological observation of live cells in the field of biology and biotechnology, or for surface observation of LSI devices in the semiconductor market (see, for example, Patent Reference 1).
Patent Reference 1: JP-A-2002-72102
FIG. 1 is a structural view of a confocal microscope described in Patent Reference 1. A video camera 1, a confocal scanner 2, a microscope 3, an actuator 4 and an objective lens 5 are arranged on the same optical axis. The confocal scanner 2 has a Nipkow disk having multiple pinholes and a microlens array associated with the disk. The confocal scanner is of a compact add-on type formed by a simple optical system and employing the Nipkow disk method.
This confocal scanner 2 is attached to a camera port of the microscope 3. Using laser beams, the confocal microscope inputs an image of a sample to the confocal scanner 2 via the objective lens 5, the actuator 4 and the microscope 3. The confocal scanner 2 acquires a confocal image of the sample and inputs it to the video camera 1.
FIG. 2 is a timing chart of various signals handled by the confocal microscope shown in FIG. 1. The video camera 1 converts the confocal image to a video signal 101 and inputs the video signal 101 to signal input terminals of the confocal scanner 2 and a synchronization interface box 9 and to a video input terminal of an image processing unit 6. The confocal scanner 2 performs rotational synchronization control of the Nipkow disk in synchronization with the video signal 101.
The synchronization interface box 9 extracts either an even-numbered pulse train or an odd-numbered pulse train of the video signal 101 to produce an internal signal A. An arbitrary waveform generator 7 generates a trigger signal 102, which is a high-level pulse signal, and then inputs the trigger signal to a trigger input terminal of the synchronization interface box 9 so that the trigger signal is used for the timing to start scanning the focal plane in question.
The synchronization interface box 9 produces an internal signal B in synchronization with the falling edge of the trigger signal 102. The internal signal B has a high-level pulse width time of approximately 35 microseconds, which is slightly longer than the time defined by the video rate of the video camera 1. The synchronization interface box 9 generates a start signal 103 by calculating the logical product of the internal signal A and the internal signal B, and inputs the start signal 103 to synchronization input terminals of the image processing unit 6 and the arbitrary waveform generator 7.
The image processing unit 6 starts capture in which the video signal 101 is converted to image data and recorded, in synchronization with the rising edge of the start signal 103 inputted from the synchronization input terminal. In accordance with the video signal 101 from the signal input terminal, the synchronization interface box 9 synchronizes all of the rotational synchronization control of the Nipkow disk by the confocal scanner 2, the timing for the image processing unit 6 to start acquiring the video signal, and the timing for an optical control system to start scanning the focal position of the objective lens.
The arbitrary waveform generator 7 starts scanning the focal position of the objective lens 5 by the optical control system in synchronization with the rising edge of the start signal 103. The arbitrary waveform generator 7 generates a scanning signal 104 and inputs it to a controller 8. The scanning signal 104 is a sawtooth signal that linearly rises from a low level to a high level over a specific period of time. The controller 8 inputs the scanning signal 104 to the actuator 4. An actuator signal 105 is a positional signal of the actual actuator. The actuator fully extends and then returns to the original position at a dash, followed by an overshoot. The period of the overshoot corresponds to a dead band.
The actuator 4 is installed between an objective lens revolver and the objective lens 5 of the microscope 3. The length of the actuator 4 in the direction of focal point of an image changes by piezoelectric driving in proportion to the level of the scanning signal 104, and thus controls the focal position of the objective lens 5. The confocal microscope acquires sliced images of the sample by scanning the focal plane in accordance with the scanning signal 104.
According to the structure described above, the rotational synchronization control of the Nipkow disk, the timing for the image processing unit to start acquiring the video signal, and the timing for the optical control system to start scanning the focal position of the lens are all synchronized with the video signal. Therefore, the positional accuracy of the confocal image is improved, thus eliminating variations in the time taken for acquiring each sliced image when acquiring multiple sliced images, and providing highly reliable sliced images.
In the case where an object is observed by using the above-described three-dimensional confocal microscope system, a phenomenon of non-uniform brightness of an image resulting from image pickup occurs even if the object has uniform brightness. One cause of such a phenomenon is shading. Shading is a phenomenon that even when an object with uniform brightness is observed, the central part of its image is bright while the peripheral part is dark because of a large quantity of light at the central part of the image and a small quantity of light at the peripheral part due to the characteristics of image pickup optical systems including a video camera.
FIGS. 3A and 3B are explanatory views for explaining the shading.
FIG. 3A shows an example of sliced image picked up by the confocal microscope.
As shown in FIG. 3A, even if an object has uniform brightness, a result of image pickup shows non-uniform distribution of brightness between the central part and the peripheral part as. FIG. 3B shows distribution of brightness of an image at a position (sectional position) indicated by a solid line in FIG. 3A. The vertical axis represents the brightness of the image and the horizontal axis represents the height of the image (distance from the center of screen). A position where the height of image is 0 (part where a chain-dotted line and a solid line intersect each other in FIG. 3A) is the central part of the sliced image. The brightness of the image is attenuated as it is away from this central part. Usually, shading correction processing is performed in order to solve the problem caused by such shading.
FIGS. 4A to 4C are explanatory views for explaining the shading correction processing.
As shown in FIGS. 4A to 4C, shading correction is to find brightness distribution (function of height of image and brightness) of an image shown in FIG. 4A picked up in advance from a sample having uniform brightness and a correction function shown in FIG. 4B, which is the reciprocal of the brightness distribution of FIG. 4A, and to multiply the correction function by the brightness of an image acquired by observing an actual observation target. Thus, the distribution of brightness becomes uniform with respect to any height of image, as shown in FIG. 4C.
However, in the conventional three-dimensional confocal microscope system, when an image of an object with uniform brightness is picked up, non-uniformity in brightness may be caused not only by the above-described shading but also by another factor. The confocal microscope is a fluorescent microscope that casts a laser beam (excited light) of a predetermined wavelength onto a sample to be observed and observes the sample by using its fluorescent wavelength. Therefore, if the sample has a certain depth (distance in the direction of optical axis) when the sample is observed by using this fluorescent microscope, the intensity of the excited light is reduced when the excited light reaches the deep part. This causes the brightness of the excited fluorescent light to be lower at the deep part than on the surface of the sample. Moreover, the fluorescent light emitted from the deep part of the sample is reduced in brightness when it reaches the surface.
In this manner, when a sample having a certain depth is observed, the brightness of fluorescent light at the deep part becomes lower than its original brightness. Even with a sample having uniform brightness, its depth causes difference in brightness of an observed fluorescent image and makes it difficult to identify the sample, which is a disadvantage.
The conventional shading correction is performed totally on a flat surface and there has been no measure to correct the brightness of an image in the direction of depth of a sample.